Molecular imprinted polymers targeting phenylalanine

ABSTRACT

Disclosed is agents and methods that target metabolism malfunctions, inborne as well as acquired, as well as methods for preparation of the agents. In particular, the invention relates to methods for preparing molecular imprinted polymers with high binding capacity for phenylalanine or tyrosine, MIPs that bind phenylalanine or tyrosine, and methods for treating phenylketonuria, alkaptonuria, and hypertyrosinemia.

FIELD OF THE INVENTION

The present invention relates to agents and methods that targetmetabolism malfunctions, inborne as well as acquired, as well as methodsfor preparation of the agents. In particular, the invention relates tomethods for preparing molecular imprinted polymers with high bindingcapacity for phenylalanine or tyrosine, MIPs that bind phenylalanine ortyrosine, and methods for treating phenylketonuria, alkaptonuria, andhypertyrosinemia.

BACKGROUND OF THE INVENTION

Previously, the present inventors have provided methods for preparationof molecular imprinted polymers (MIPs) that provide for hitherto unseencapacities for binding by the resulting MIP compositions. In this way,the inventors have provided novel treatments of certain metabolismmalfunctions that it has not previously been possible to address withmedicinal products: for instance, treatment of phenylketonuria by dailyadministration of high-capacity MIPs that confine phenylalanine to thegastrointestinal tract has been a major advance. Another advantage ofthe improved MIPs obtained by these methods are their use in highlysensitive assays.

The technologies that found the basis of the present invention are e.g.disclosed in WO 2007/095949, WO 2011/033021, WO 2013/127433, andPCT/EP2017/056059, the contents of which are all incorporated herein byreference.

OBJECT OF THE INVENTION

It is an object of embodiments of the invention to provide for improvedagents for treatment of errors of metabolism. An object of otherembodiments is to provide novel methods for preparing such improvedagent. And, an object of yet other embodiments of the invention is toprovide novel treatments of phenylketonuria (PKU), alkaptonuria, andhypertyrosinemia.

SUMMARY OF THE INVENTION

It has been found by the present inventor(s) that a carefully selectedmixture of monomers and a target provides for MIPs with outstandingproperties in terms of their binding capacity and specificity when theMIPs are purified according to the scheme disclosed herein. The MIPsobtained this way are believed to be novel chemical entities.

It has also been found by the present inventors that MIPs prepared usinga Phe-containing template provide for a subpopulation of MIPs thatsurprisingly bind L-tyrosine (Tyr) with high affinity, meaning that onepolymerization process surprisingly leads to two distinct products withvery different but useful binding properties.

Finally, it has been found by the present inventors that the Phe-bindingMIPs of the present invention have a binding capacity of such amagnitude that effective treatment of PKU in a standard adult person (70kg) that ingests a diet containing recommended (by the authorities)amounts of protein can be attained by administration of less than 35 gMIP per day dependant on the proportional protein intake.

So, in a first aspect the present invention relates to a method for thepreparation of molecular imprinted polymer (MIPs), which specificallybind L-phenylalanine (Phe), said method comprising the steps of

a) polymerization of a mixture comprising

-   -   2-methylprop-2-enoic acid (MAA),    -   1,4-bis(acryloyl)piperazine/1,4-diacryloylpiperazine (DAP), and    -   a template molecule consisting of L-Phe or a L-Phe derivative        exposing a phenylalanine motif        in the presence of a catalyst and an oxidizing agent,        so as to obtain a cross-linked imprinted polymer,        b) if necessary (that is, if particle sizes after polymerization        does not include a sufficient amount of MIPs having particle        sizes smaller than 63 μm) subsequently fragmenting the        cross-linked imprinted polymer obtained in step a) to obtain a        first fragmented polymer, and collecting the MIPs having        particle sizes smaller than 63 μm,        c) optionally washing and drying the polymer fraction obtained        from step b,        d) fragmenting further the polymer fraction obtained from step c        and collecting a second fragmented polymer having particle sizes        in the range 150-250 nm,        e) subjecting second fragmented polymer obtained from step d to        affinity chromatography where Phe constitutes the affinity tag        (optionally as part of a larger molecule) in a chromatographic        matrix, and        f) recovering MIPs binding to Phe in step e.

In a second aspect, the invention relates to a molecular imprintedpolymer (MIP), which specifically binds L-phenylalanine (Phe), whereinsaid MIP is comprised of polymerized methacrylic acid (MAA) cross-linkedwith 1,4-diacryloylpiperazine (DAP).

In a third aspect, the invention relates to a composition comprising theMIP of the second aspect of the invention and embodiments thereof, saidcomposition comprising a pharmaceutically acceptable carrier and/ordiluent and/or excipient, wherein said composition is adapted for oraladministration.

In a fourth aspect, the invention relates to a method for thepreparation of MIPs, which specifically bind L-tyrosine (Tyr), saidmethod comprising recovering MIPs that bind L-tyrosine from an initialcomposition of MIPs that have been prepared using, as the templatemolecule during polymerization and cross-linking, a molecule comprisingat least one phenylalanine motif and comprising no Tyr residues.

In a fifth aspect, the invention relates to a molecular imprintedpolymer (MIP), which specifically binds L-tyrosine (Tyr), wherein saidMIP is comprised of polymerized methacrylic acid (MAA) cross-linked with1,4-diacryloylpiperazine (DAP).

In a sixth aspect, the invention relates to a composition comprising theMIP of the fifth aspect of the invention and embodiments thereof, saidcomposition comprising a pharmaceutically acceptable carrier and/ordiluent and/or excipient, wherein said composition is adapted for oraladministration.

In a seventh aspect, the invention relates to a method of treatment ofphenylketonuria in a person in need thereof, said method comprisingadministering to a person in need thereof the MIPs according to thesecond aspect and embodiments thereof or the composition of the thirdaspect and embodiments thereof so as to deliver a daily effective doseof MIPs of the second aspect.

Finally, in an eighth aspect, the invention relates to a method fortreatment of tyrosineamia and/or alkaptonuria, the method comprisingadministering to a person in need thereof

1) MIPs that bind phenylalanine, preferably the MIPs of the secondaspect or the composition of the third aspect so as to deliver a dailyeffective dose of MIPs according to any one of claims; and/oradministering2) MIPs of the fifth aspect and embodiments thereof or the compositionof the sixth aspect and embodiments thereof, so as to deliver a dailyeffective dose of MIPs of the fifth aspect and embodiments thereof.

LEGENDS TO THE FIGURE

FIG. 1: Effect of Phe binding MIP administration to PKU mice on Pheblood concentration. Each group contained 5 PKU mice, that were held ona phenylalanine restricted diet until start of the study. Each mouse wasdosed 3 times a day for 2 days with either 20.7 mg protein per dosage or20.7 mg protein per dosage plus Phe binding MIPs. A blood sample wasdrawn from each mouse prior to first dosage and 2 h after last dosageand analysed for phenylalanine. Three studies were performed withdifferent dosages. Dotted lines/triangle data points representmeasurements on animals receiving protein only; solid lines/square datapoints represent measurments on animals receiving both protein and Phebinding MIPs (“phelimin”).

DETAILED DISCLOSURE OF THE INVENTION Definitions

A “molecular imprinted polymer” (MIP) is a polymer comprising cavities(or voids) that at least in part correspond to one or more templatemolecules that have been incorporated in a monomer matrix includingcross-linking monomers prior to polymerization. The resulting polymerafter polymerization includes a number of cavities which correspond inshape to the template molecule. Typically the MIP is sequestered(fragmented, micronized) into small particles, thereby facilitatingremoval of template and leaving partial cavities open for interactionwith a target molecule which resembles or is identical to the templatemolecule. In the present specification and claims, the term MIPgenerally refers to the particulate form of a MIP, meaning that theterms “MIP” and “MIPs” are used interchangeably with the expressions MIPparticle and MIP particles, respectively.

It will be understood that the MIPs employed in the present inventionare insoluble molecules/entities even though they may appear stable insuspension if sufficiently small. The MIPs are especially suitable aspharmaceutical for use in the gastrointestinal tract since theirinsolubility limits or prevents their passage into the body (e.g. intocirculation) from the gastrointestinal tract. In other words, whenadministered orally, the MIPs used in the present invention willsubstantially remain confined to the gastrointestinal tract until theyare disposed off in the faeces.

A “raw MIP” is a MIP which has not yet been subjected to affinitypurification and hence contains a heterogenic mixture of MIPs withdifferent binding characteristics, e.g. even MIPs with no ascertainablebinding to the template molecule.

“Micronization” and “fragmentation” (used interchangeably) denote theprocess of sequestering MIPs which may still contain template intosmaller particles. Any method suitable for this purpose may be used, cf.below.

A “target molecule” is in the present context any molecule or molecularmotif to which a MIP can bind.

A “template molecule” is normally identical to the target molecule, butmay also be a mimic thereof (i.e. a molecule having at least in part anidentical 3D structure and profile which matches that of the targetmolecule—a mimic may for instance be constituted by a fragment of thetarget molecule or as in the present application by a larger molecule ofwhich the intended target is an important part). The template serves asthe “generator” of the voids in the MIP structure which subsequently areto be able to bind the target molecule.

A “phenylalanine-derivative where a phenylalanine motif is exposed”denotes a template molecule comprising a benzyl group, a phenyl ring orthe benzyl group or phenyl ring combined with either a carboxylic groupor an amino group. Therefore, any peptide containing phenylalanine(preferably di- or tripeptides) can be used as such a template molecule.

“Affinity purification” denotes any method for purification of asubstance where specific binding between the substance and a bindingpartner is utilised. Many such methods utilise a capture agent bound toa solid support (such as a chromatographic matrix) which catches thesubstance. Typical examples known in the art are affinity purificationusing antibodies as capture agents coupled to chromatographic beads forpurifying antigens that bind the antibody. It will be understood thatthe affinity purification methods applied according to the presentinvention are those which are capable of capturing suspended insolubleMIP particles having the sizes discussed herein.

A “solid phase” is in the present context any material which may be usedto anchor a capture agent by means of covalent or non-covalent binding.Hence, any material (plastic polymers, sugars, metals, glass, silica,rubber etc) which is conventionally used in the preparation ofchromatographic materials may serve as the solid phase. The solid phasematerial may contain suitable functional groups which allow coupling ofthe capture agent to the material in question. Such derivatizedmaterials are known to the person of skill in the art of chromatographicpurification of proteins and other macromolecules. Further, the solidphase may have any physical form which allows for capture of relativelylarge and insoluble particles such as MIPs (when comparing with singlebiomolecules such as proteins). Hence, the solid phase may be in theform of fibers (preferably hollow), a chromatography matrix, beads(preferably those that may be separated by electromagnetic means) or anyother suitable form, cf. below.

When discussing sizes of MIPs herein, e.g. MIPs being smaller or largerthan a given length X (e.g. 63 μm), is herein meant that the size of theparticles are such that they are capable or incapable of passing throughsieves having a defined cut-off, i.e. diameter of the holes in the sieveX—the particles that pass through are “smaller than” X, and theparticles that are retained are “larger than” X. In other words, amolecule may in theory be larger than 63 μm along an axis, but stillcapable of passing through a 63 μm sieve: in such a case, the moleculeis said to be smaller than 63 μm.

Certain abbreviations are used in the present disclosure: L-Phe denotesL-phenylalanine, Gly denotes glycine, Ala denotes either L-alanine,D-alanine, or a mixture of both, L-Asp denotes L-aspartic acid, Medenotes methyl, and OMe denotes O-methyl (—O—CH₃).

Specific Embodiments of the Invention Embodiments Relating to the 1^(st)Aspect of the Invention

As indicated above, the first aspect relates to a method for thepreparation of molecular imprinted polymer (MIPs), which specificallybind L-phenylalanine (Phe), said method comprising the steps of

a) polymerization of a mixture comprising

-   -   2-methylprop-2-enoic acid (MAA),    -   1,4-bis(acryloyl)piperazine (DAP), and    -   a template molecule consisting of L-Phe or a L-Phe derivative        exposing a phenylalanine motif in the presence of a catalyst and        an oxidizing agent,        so as to obtain a cross-linked imprinted polymer,        b) if necessary (due to large particle sizes) subsequently        fragmenting the cross-linked imprinted polymer to obtain a first        fragmented polymer and collecting the fraction thereof having        particle sizes smaller than 63 μm,        c) optionally washing and drying the polymer fraction obtained        from step b,        d) fragmenting further the polymer fraction obtained from step c        and collecting a second fragmented polymer having particle sizes        in the range 150-250 nm,        e) subjecting second fragmented polymer obtained from step d to        affinity chromatography where Phe constitutes the affinity tag        in a chromatographic matrix, and        f) recovering MIPs binding to Phe in step e.

The polymerization mixture in step a preferably contains MAA and DAP ina MAA:DAP molar ratio of 5-30. Preferably, the ratio is between 6 and27, such as between 7 and 24, 8 and 21, 9 and 18, and preferably between10 and 15.

The exact polymerization method can vary. As will be clear from theexamples, equally good results have been obtained using bulkpolymerization followed by micronization step and using the reversephase emulsion polymerization method in example 4. The latter methodprovides for MIP particles of such a generally small size thatmicronization in step b is not relevant, meaning that simple collectionof sufficiently small polymer particles can be carried out directly instep b, see below.

The preferred molar ratio MAA:template molecule is between 1.0 and 4.0.Values can vary in this interval, meaning that the ratio can be selectedfrom about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6,about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9,about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about3.6, about 3.7, about 3.8, about 3.9, and about 4.0.

The template molecule is preferably an L-Phe derivative in the form of apeptide containing at least one L-Phe residue, which is a typical formof a L-Phe derivative exposing a phenylalanine motif. Good results havebeen obtained with L-Phe containing dipeptides or tripeptides, which arethus particularly preferred template molecules. Such di- or tripeptidescan e.g. be selected from the group consisting of Gly-L-Phe(glycyl-L-phenylalanine), Ala-L-Phe, L-Asp-L-Phe-OMe, L-Asp-L-Phe,Gly-Gly-L-Phe, Ala-Gly-L-Phe, Gly-Ala-L-Phe, L-Phe-Gly, L-Phe-Ala,L-Phe-L-Phe, L-Phe-Ala-L-Phe, and L-Phe-Gly-L-Phe. A particularlypreferred template molecule is Gly-L-Phe.

The catalyst in step a is typically selected from those that catalysepolyacrylamide gel polymerization in aqueous media. Useful catalysts aretetramethylethylenediamine (TEMED) and dimethylpiperazine. The preferredcatalyst is TEMED. These catalysts typically have to be used togetherwith a strong oxidizing agent. Hence, it is preferred that the oxidizingagent in step a) is selected from the group consisting of ammoniumpersulfate, potassium persulfate, and sodium thiosulfate, preferablyammonium persulfate (APS).

The fragmentation method utilised in step b is not essential, and isonly applied if the particle sizes of the molecular imprinted polymersobtained from step a are too large. Essentially any method fordownsizing of MIPs known in the art can be used: for instance, thefragmentation in step b can comprise grinding, milling, explosion,hammering, ball milling, cryo grinding, or collision homogenisation aswell as any combination of these methods.

In order to ensure that the MIP particles obtained in step b) aresmaller than 63 μm, standard metal sieves with defined cut-offs can beused. For instance, it is preferred that the MIPs collected in step bhave sizes in the 25-63 μm range and this can be attained by utilisingsieves with a 63 μm cut-off, and collecting the MIPs that are able topass through. Subsequently, sieves with a 25 μm cut-off can be used,where the material retained is used in subsequent steps. However, sincesmaller particles can be subjected to the subsequent steps, step b neednot isolate particles of any defined minimum size.

The optional washing is performed at alternating pH with an organicsolvent, e.g. as shown in the examples. This ensures that accessiblebound template is effectively removed prior to the following steps. Oneconvenient way of washing the MIPs is to pack them in a column such asan HPLC column, thereby facilitating washing under elevated pressurewith a solvent passing through the HPLC column.

Subsequent step d (the further fragmentation of MIP particles) isconveniently carried out in a ball mill or bead mill but as for step bthe exact micronization method employed in inessential for the result tobe achieved as long as the method is capable of providing a sufficientfragmentation. Hence, the methods recited above as convenient in step bare all relevant for the purposes of step d.

After step d, the MIP particles are now of such a small size that theyremain in suspension for a prolonged time. To isolate/collect the MIPsin step d can hence be carried out by suspension of MIPs in a solvent(preferably an aqueous solvent such as water), subsequent incubation inan ultrasound bath (which can in its own right effect micronization),centrifugation, and isolation of the supernatant, which contains the150-250 nm MIPs. Also, a washing step after step d and prior to step ecan conveniently be carried out in order to remove any residualtemplate, e.g. template that has been made accessible due to the furtherdownsizing; this washing is typically carried out as a dialysis step,but also ultracentrifugation is a possibility—the washing step merelyhas to fulfil the purpose of separating MIPs and template.

The final step e and f resemble traditional chromatographic procedurescarried out on truly soluble material: the affinity chromatography instep e) is conveniently carried out on a packed bed chromatographiccolumn using a stationary chromatographic matrix, which carries thecapture probe that includes Phe) and where the MIPs are suspended in abuffered aqueous solvent. Finally, the MIPs are recovered from thecolumn by elution methods known per se. As shown in the examples, thepreferred elution liquid is ethanol in water, such a 40% ethanol.

Embodiments Relating to the 2^(nd) Aspect of the Invention

The molecular imprinted polymer (MIP) of the invention, whichspecifically binds L-phenylalanine (Phe), wherein said MIP is comprisedof polymerized methacrylic acid (MAA) cross-linked with1,4-diacryloulpiperazine (DAP), is believed to be a novel chemicalentity. The molar ratio between the MAA and DAP monomer residues ispreferably between 5 and 30. However, the ratio is preferably between 6and 27, such as between 7 and 24, 8 and 21, 9 and 18, and preferablybetween 10 and 15. In particular, it is preferred that this MIP of theinvention is obtainable or obtained by the method of the first aspect ofthe invention.

As discussed above, the MIP of the second aspect and the MIP obtained bymeans of the method of the first aspect exhibit a very high bindingcapacity and affinity for its target, L-Phenylalanine. The MIP has aK_(D) for binding to Phe of less than 10⁻⁷, but lower values have beenmeasured: less than 10⁻⁸, less than 9×10⁻⁹, less than 8×10⁻⁹, less than7×10⁻⁹, less than 6×10⁻⁹, less than 5×10⁻⁹, less than 4×10⁻⁹, less than3×10⁻⁹, and less than 2×10⁻⁹. A preferred K_(D) of about 10⁻⁹ has beenmeasured with the L-Phe binding MIPs disclosed herein.

Embodiments Relating to the 3^(rd) Aspect of the Invention

The composition of the 3^(rd) aspect of the invention is preparedaccording to conventional formulation techniques. Since the activeprinciple has an almost infinite shelf-life, the MIPs can beincorporated into any common oral dosage form: tablets, capsules,powders, granules, medicated gums, and suspensions, where all otheringredients (preservatives and other excipients) are standard choices.

However, in order to avoid problems with growth of fungi, bacteria oralgae, a pharmaceutically acceptable disinfectant or preservative can beincluded in those formulations where such growth could present aproblem.

The MIPs may also be formulated so that they are or can be incorporatedinto foods and drinks. Since it has turned out that the MIPs of theinvention exhibit no taste, their inclusion in any kind of ingestibleproduct has proven to be unproblematic.

For details of preparation of compositions of the invention for oralintake, reference is generally made to Remington's “Essentials ofPharmaceutics”, Pharmaceutical Press 2012 (published 2013), ISBN 978 085711 105 0, in particular chapters 30, 31, and 32.

Embodiments Relating to the 4^(th) Aspect of the Invention

As indicated above, it has surprisingly turned out the imprintingprocess described above in the first aspect of the invention providesfor a sub-population of MIPs that are strong binders of the amino acidL-tyrosine. This is in spite of the fact that the template moleculeGly-L-Phe, which has been tested, does not include any Tyr functionalityand in spite of the fact that MIPs obtained by the method of the firstaspect (after purification) are highly specific for L-phenylalanine ande.g. binds significantly less to D-phenylalanine or approx. 700 timesless effectively to L-Tyr. This evidences that preparation of “raw” MIPsfrom polymerization with a Phe-containing template provides forTyr-binding MIPs also. So, the broadest embodiment of the 4^(th) aspectrelates as indicated above to a method for the preparation of MIPs,which specifically bind L-Tyrosine (Tyr), said method comprisingrecovering MIPs that bind L-Tyrosine from an initial composition of MIPsthat have been prepared using, as the template molecule duringpolymerization and cross-linking, a molecule comprising at least onephenylalanine motif and comprising no Tyr residues. In particular, thetemplate molecule is preferably a template molecule discussed in thecontext of the first aspect of the invention; as demonstrated in theExample section, this provides for a very effective imprinting process.

It is also preferred that the initial composition of MIPs is separatedin at least 2 fractions, and recovering Tyr binding MIPs from afraction, which is essentially free from MIPs that bind thePhe-containing capture probe—this may e.g. be accomplished by firstseparating the MIPs that bind L-Phe or Phe motifs with high affinity.The Expression “Phe motif” is in the present context typically meant aL-Phe residue such as an internal, N- or C-terminal Phe residue in apeptide or polypeptide.

So, a preferred embodiment entails the steps of

a) polymerization of a mixture comprising

-   -   2-methylprop-2-enoic acid (MAA),    -   1,4-bis(acryloyl)piperazine (DAP), and    -   a template molecule discussed in the context of the first aspect        of the invention (preferably Gly-L-Phe)    -   in the presence of a catalyst and an oxidizing agent, so as to        obtain a cross-linked imprinted polymer,        b) if necessary (cf. above) subsequently fragmenting the        cross-linked imprinted polymer to obtain a first fragmented        polymer and collecting the fraction thereof having particle        sizes smaller than 63 μm,        c) optionally washing and drying the polymer fraction obtained        from step b,        d) fragmenting the polymer fraction obtained from step c and        collecting a second fragmented polymer having particle sizes in        the range 150-250 nm, and        e) subjecting the second fragmented polymer obtained from step d        to affinity chromatography where    -   e1) Tyr constitutes the affinity tag in a chromatographic        matrix, or    -   e2) Phe constitutes the affinity tag in a chromatographic        matrix, recovering MIPs not binding to Phe, and subjecting the        MIPs not binding to Phe to further affinity chromatography where        Tyr constitutes the affinity tag (optionally as part of a larger        molecule) in a chromatographic matrix, and        f) recovering MIPs binding to Tyr in step e.

As is clear from this embodiment, steps a-d are identical with steps a-din the first aspect of the invention. Hence, all features relating toprocess parameters and reagents disclosed above for these steps of thefirst aspect of the invention apply mutatis mutandis to steps a-d of the4^(th) aspect.

Likewise, the remaining steps also can be carried out according to thegeneral teachings provided above in respect of the first aspect. Whenthe route e1 is selected, no step of removing Phe-binding MIPs isperformed, so potentially the MIPs obtained this way may includePhe-binders—if route e2 is followed, the steps up to the Tyr affinitychromatography are identical with the steps prior to Phe purification inthe first aspect, and all disclosures relating to these of the firstaspect apply mutatis mutandis to the fourth aspect.

The final step e and f further resembles traditional chromatographicprocedures carried out on truly soluble material: the affinitychromatography using Tyr as affinity tag in step e) is convenientlycarried out on a packed bed chromatographic column using a stationarychromatographic matrix, which carries the capture probe that includes oris L-Tyr) and where the MIPs are suspended in a buffered aqueoussolvent. Finally, the MIPs are recovered from the column by elutionmethods known per se. As shown in the examples, the preferred elutionliquid is ethanol in water, such a 40% ethanol.

Embodiments Relating to the 5^(th) Aspect of the Invention

It is believed that the molecular imprinted polymer (MIP), whichspecifically binds L-tyrosine (Tyr) described for the 5^(th) aspect is anovel chemical entity. Its basic chemical composition is identical withthat of the MIP of the second aspect of the invention, meaning that themolar ratio between MAA residues and DAP residues preferably is between5 and 30. However, the ratio is preferably between 6 and 27, such asbetween 7 and 24, 8 and 21, 9 and 18, and preferably between 10 and 15.In particular, it is preferred that this MIP of the invention isobtainable or obtained by the method of the fourth aspect of theinvention.

The K_(D) values for binding between the Tyr-binding MIPs and L-Tyr arepreferably of the same values as those discussed above for Phe-bindingMIPs that bind to L-Phe.

Embodiments Relating to the 6^(th) Aspect of the Invention

The composition of the 5^(th) aspect of the invention is preparedaccording to conventional formulation techniques. Since the activeprinciple has an almost infinite shelf-life, the MIPs can beincorporated into any common oral dosage form: tablets, capsules,powders, granules, medicated gums, and suspensions, where all otheringredients (preservatives and other excipients) are standard choices.

However, in order to avoid problems with growth of fungi, bacteria oralgae, a pharmaceutically acceptable disinfectant or preservative can beincluded in those formulations where such growth could present aproblem.

The MIPs may also be formulated so that they are or can be incorporatedinto foods and drinks. Since it has turned out that the MIPs of theinvention exhibit no taste, their inclusion in any kind of ingestibleproduct has proven to be unproblematic.

For details of preparation of compositions of the invention for oralintake, reference is generally made to Remington's “Essentials ofPharmaceutics”, Pharmaceutical Press 2012 (published 2013), ISBN 978 085711 105 0, in particular chapters 30, 31, and 32.

Embodiments Relating to the 7^(th) Aspect of the Invention

The MIPs of the second aspect, as well as the composition of the 3^(rd)aspect of the invention are useful for treatment of diseasescharacterized by excess blood concentrations of L-Phe. As such a methodof treatment of phenylketonuria in a person in need thereof, said methodcomprising administering to a person in need thereof the MIPs of thesecond aspect or the composition of the third aspect is provided. Thismethod may e.g. be carried out as is generally disclosed in WO2011/033021.

As discussed above, it is possible to control Phe in persons sufferingfrom PKU by administering a feasible dosage per standard meal of theMIPs of the second aspect: an effective dose per meal is preferably 1-35g/70 kg (i.e. 1-35 grams per 70 kg of bodyweight). Preferably, theeffective dose per meal is at most or about 34 g/70 kg, such as at mostor about 33, at most or about 32, at most or about 31, at most or about30, at most or about 29, at most or about 28, at most or about 27, atmost or about 26, at most or about 25, at most or about 24 at most orabout 23, at most or about 22, at most or about 21, at most or about 20,at most or about 19, at most or about 18, at most or about 17, at mostor about 16, at most or about 15, at most or about 14, at most or about13, at most or about 12, at most or about 11, at most or about 10, atmost or about 9, at most or about 8, at most or about 7, at most orabout 6, at most or about 5, at most or about 4, at most or about 3, atmost or about 3, and at most or about 2 g/70 kg. As shown in theexamples, a dose per meal of about 11 g/70 kg bodyweight has until nowbeen found to be effective.

So, a preferred range for the effective daily dosage (comprising 3meals) is 3-105 g per 70 kg body weight, with more narrow dosageintervals being 20-45 g/70 kg, such as 25-40 g/70 kg, and 30-35 g/kgbodyweight. Hence, the daily effective dose may be about 4, about 5,about 6, about 7, about 8, about 9, about 10, about 11, about 12, about13, about 14, about 15, about 16, about 17, about 18, about 19, about20, about 21, about 22, about 23, about 24, about 25, about 26, about27, about 28, about 29, about 30, about 31, about 32, about 33, about34, about 35, about 36, about 37, about 38, about 39, about 40, about41, about 42, about 43, about 44, about 45, about 46, about 47, about48, about 49, about 50, about 51, about 52, about 53, about 54, about55, about 56, about 57, about 58, about 59, about 60, about 61, about62, about 63, about 64, about 65, about 66, about 67, about 68, about69, about 70, about 71, about 72, about 73, about 74, about 75, about76, about 77, about 78, about 79, about 80, about 81, about 82, about83, about 84, about 85, about 86, about 87, about 88, about 89, about90, about 91, about 92, about 93, about 94, about 95, about 96, about97, about 98, about 99, about 100, about 101, about 102, about 103,about 104, and about 105 g/70 kg.

As will be understood from the above, the MIPs are administered orally,typically as a suspension, as part of a food or drink, or as a solidformulation.

Embodiments Relating to the 8^(th) Aspect of the Invention

In analogy to the 7^(th) aspect is provided a method for treatment oftyrosineamia and/or alkaptonuria, the method comprising administering toa person in need thereof 1) MIPs that bind phenylalanine, preferably theMIPs of the second aspect and embodiments thereof or the composition ofthe 3^(rd) aspect and embodiments thereof so as to deliver a dailyeffective dose of MIPs according to any one of claims of the secondaspect; and/or 2) MIPs that bind tyrosine of the 5^(th) aspect or thecomposition of the 6^(th) aspect, so as to deliver a daily effectivedose of MIPs of the 5^(th) aspect and embodiments thereof.

In preferred embodiments, this is done as adjuvant therapy tonitisionone treatment of alkaptonuria, see below in Example 3.

As discussed above, it is possible to control Tyr in persons sufferingfrom PKU by administering a feasible daily dosage of the Phe and/or Tyrbinding MIPs: an effective dose is preferably

1-35 g/70 kg (i.e. 1-35 grams per 70 kg of bodyweight). Preferably, thedaily effective does is at most or about 34 g/70 kg, such as at most orabout 33, at most or about 32, at most or about 31, at most or about 30,at most or about 29, at most or about 28, at most or about 27, at mostor about 26, at most or about 25, at most or about 24 at most or about23, at most or about 22, at most or about 21, at most or about 20, atmost or about 19, at most or about 18, at most or about 17, at most orabout 16, at most or about 15, at most or about 14, at most or about 13,at most or about 12, at most or about 11, at most or about 10, at mostor about 9, at most or about 8, at most or about 7, at most or about 6,at most or about 5, at most or about 4, at most or about 3, at most orabout 3, and at most or about 2 g/70 kg.

So, a preferred range for the effective daily dosage is 10-15 g/70 kg,such as about 10, about 11, about 12, about 13, about 14 or about 15g/70 kg.

As will be understood from the above, the MIPs are administered orally,typically as a suspension, as part of a food or drink, or as a solidformulation.

Example 1 Preparation of Phe Binding MIPs

Polymer is synthesized as a bulk polymer, also known as a monolith. Themonomers, MAA (2-Methylprop-2-enoic acid, CAS no 79-41-4) and DAP(1,4-Bis(acryloyl)piperazine, CAS no 6342-17-2), as well as thetemplate, the dipeptide Gly-L-Phe (GF), are dissolved in water in aglass tube and degassed by bubbling an inert gas through the solution.APS (ammonium persulfate, CAS No. 7727-54-9) and TEMED(tetramethylethylenediamine, CAS No. 110-18-9) are added and the glasstube is placed in a water bath at elevated temperature and left forpolymerization overnight.

The glass tube is mechanically broken and the polymer, approx. 10 g, iscollected and subject to a first down-sizing in a rotor mill. Thedown-sized polymer is sieved and the 25-63 μm fraction is collected andwashed thoroughly packed in an HPLC column under high pressure usingalternating pH values and organic solvents.

The washed and dried polymer is then subject to a second down-sizing byball-milling in a ball-mill with planetary motion. From the ball-milledsample a suspendable fraction is harvested by suspending the ball-milledpolymer in water, incubate in an ultrasound bath and finally centrifuge.The supernatant contains a suspension of polymer particles with a sizeof approx. 150-250 nm. The polymer suspension is buffered with a 10×PBSbuffer stock, pH 8.0, and is then ready for chromatography.

The suspension of polymer is applied to an affinity chromatographycolumn, with PBS as running buffer, where Phe has been immobilized onthe chromatography matrix as affinity tag. When the polymer sample haspassed, the column is washed to remove polymer particles not tightlybound to the column and affinity captured polymer particles aresubsequently eluted by changing the running buffer by including anorganic solvent.

The eluted polymer particles are transferred into water or PBS by e.g.by dialysis or tangential flow filtration and concentrated before finalformulation.

In a specific example, the entire general procedure was carried out asfollows.

1. Pre-polymerization: 1.667 g Gly-L-Phe and 50 ml water is mixed andstirred in a 100 ml flask for 2 min and 1.301 g MAA is added. After 2min of further stirring and 2 min in an ultrasound bath (Branson 2510)at room temperature (RT) in order to fully dissolve Gly-L-Phe, 38.834 gDAP is added. The flask is placed at 40° C. in a water bath withcontinued stirring for 5 min to achieve a homogenous solution. The flaskis then left at RT for 1-2 h.2. Polymerization: 17 ml of the pre-polymerization solution istransferred to a culture glass tube (25×150 mm) containing 100 mg APS,vortexed for 1 min (2,500 rpm) and placed in an ultrasound bath for 5min. The solution is sparged for 5 min with argon, 75 μl of a 25%solution in water freshly prepared TEMED is added and the tube isvortexed for 20 s at 2,500 rpm. The tube is closed with a tight cap andimmediately placed at 70° C. in water bath and left for polymerizationovernight.3. Downsizing: The polymer is manually broken into approximately 1 cmpieces and subsequently milled in a rotor-mill (Fritsch Pulverisette 14)supplied with a 500 μm sieving ring at 13,000 rpm.

The milled polymer is sieved (dry) through 63 and 25 μm stainless sievesfor isolation of the 25-63 μm fraction. The isolated the 25-63 μmfraction is suspended in acetone and left for sedimentation for 5-10min.

The supernatant is removed, and the suspension/sedimentation/decantingcycle is repeated until the supernatant appears clear. The settledpolymer is dried overnight at 60° C. The dry polymer is packed into anHPLC column and washed under elevated (5-15 bar) pressure with thefollowing solvents:

-   -   A. Ethanol/acetic acid 4:1 (vol/vol); 540 ml    -   B. Ethanol; 100 ml    -   C. Ethanol/NaOH (5M)/H₂O 5:2:3; 200 ml    -   D. Ethanol; 100 ml    -   E. Ethanolacetic acid/H₂O 18:1:1; 200 ml    -   F. Ethanol; 300 ml    -   G. Acetone; 100 ml

The washed polymer is dried overnight at 60° C. and ball milled in aFritsch Pulverisette 7 in a ZrO₂ milling chamber using ZrO₂ balls in twosteps; using 1.5 mm balls in the first step and 0.5 mm balls in thefinal step. In both steps 5 g balls is placed in a 12 ml milling chamberand 1 g polymer is placed on top of the balls. Milling schedule is 30min milling at 750 rpm, 30 min pause, 30 min milling at 750 rpm inreverse mode, etc. 10 cycles per step.

4. Harvest of a stable, suspendable fraction: After milling, the polymeris suspended in approximately 30 ml water, separated from the millingballs by a pipette, transferred to a 50 ml polypropylene centrifuge tubeand placed in an ultrasound bath (Branson 2510) for approx. 30 min.After centrifugation at 4,500 G for 15 min the supernatant, around 25ml, is harvested and contains a stable suspension of polymer particleswith a size range of 150-250 nm. The absorbance at 254 nm in a 1 cmquartz cuvette is around 500-1,500 AU.5. Preparation of sample for chromatography: The harvested fraction isdialyzed by Tangential Flow Filtration (TFF) using a Polyethersulfonefilter with a 30.000 Dal cut off. The polymer suspension is subsequentlybuffered by 10×PBS (phosphate buffered saline) and diluted in 1×PBS (pH8.0, 20 mM phosphate, 150 mM NaCl) to a final polymer concentration of30 AU at 254 nm.6. Isolation of Phe binding MIPs by affinity chromatography and work-up:The TFF'ed polymer is run on an affinity chromatography column (HiTrap®NHS-activated 5 ml column, GE Healthcare, 17-0717-01) coupled withGly-L-Phe following the manufacturer's recommendations. 10 ml of thesample is passed through the column at 1 ml/min. The column is washedwith additional 50 ml at 1 ml/min and the column is then eluted with 40%ethanol in water at 1 ml/min. The running buffer is PBS, pH 8.0. Theeluted fraction of MIPs is dialyzed into water and concentrated by TFFand optional freeze dried.

Example 2

Isolation of Tyrosine (Tyr) Binding MIPs from the GlyPhe ChromatographyRun-Through

The run-through from the from the process for isolating Phe binding MIPs(affinity chromatography using a Gly-L-Phe coupled column) in step 6 inExample 1 is passed over a column (HiTrap® NHS-activated 5 ml column, GEHealthcare, 17-0717-01) coupled with Tyr following the manufacturer'srecommendations. The same protocol is used as for running the Gly-L-Phecoupled column except that 20 ml sample (run through from the isolationof Phe binding MIPs) is applied. The column is washed with additional 50ml at 1 ml/min and the column is then eluted with 40% ethanol in waterat 1 ml/min. The running buffer is PBS, pH 8.0. The eluted fraction ofMIPs is dialyzed into water and concentrated by TFF and finally freezedried.

Surprisingly, this provides for isolation of high-capacity, high-avidityTyr binding MIPs.

Patients suffering from either Tyrosinemia or Alkaptonuria and who aretreated with nitisionone (CAS no 104206-65-7) are known to accumulateunhealthy amounts of tyrosine in the blood as a side effect of theirtreatment.

The only currently known remedy for this side effect is to prescribe aprotein restricted diet. Since both Tyrosinemia and Alkaptonuriapatients exhibit normal conversion of phenylalanine to tyrosine, i.e.the surplus of tyrosine is derived from dietary tyrosine as well ashydroxylated phenylalanine, a reduction in dietary phenylalanine uptakewith Phe binding MIPs is considered beneficial for these patients.Furthermore, these patients should benefit from a reduction in dietarytyrosine by treatment with tyrosine binding MIPs in a manner analogousto the treatment of PKU patients with Phe binding MIPs.

Example 3 Establishing Human Doses of Phe-Binding MIPs in a Mouse Model

Each study group contained 5 PKU mice that were held on a phenylalaninerestricted diet until start of the study.

Each mouse was administered 3 times a day over the course of 2 days witha dose of either 20.7 mg protein per dosage or 20.7 mg protein perdosage plus Phe-binding MIPs of the present invention.

A blood sample was drawn from each mouse prior to first dosage and 2 hafter last dosage and analysed for L-phenylalanine. Three studies wereperformed with different dosages of Phe binding MIPs (see FIG. 1;dosages were 21, 18, and 13 mg MIP).

In a PKU mouse model (which is deficient in phenylalanine hydroxylase)is was found (FIG. 1C) that a ratio of Phe binding MIPs to protein ratio(w/w) of 0.63 (corresponding to 13 mg Phe binding MIPs per dose)neutralized the Phe from the protein administered to the mice, thusleading to a Phe concentration in the blood that was the same after 3dosages per day for 2 days in the MIPs treated mice. On the other hand,mice receiving the same amount of protein, but no Phe-binding MIPs,exhibited an increase of Phe blood concentration of approximately 140 μMover the course of the 2 day study.

If larger doses of Phe binding MIPs were administered to the mice, i.e.increasing the MIPs to protein ratio, the treated mice exhibited adecrease in blood Phe concentration after 2 days of treatment (FIGS. 1Aand 1B)

If the MIPs to protein ratio from the mouse studies is transferred tohuman PKU patients it means that a standard meal (0.75 g protein/kgbodyweight/day) in a 70 kg person would require 11 g Phe Binding MIPs ofthe present invention to neutralize the Phe obtained from the dietaryprotein. In case the patient wants to decrease the blood Phe, butconsume the same amount of protein, a higher Phelimin to protein ratiocould be used.

Example 4 Industrial Scale Synthesis

Synthesis and processing of the polymers of the present invention in anindustrial format, such as reverse phase emulsion polymerization, istypically performed through the steps described in the followingexample, which included the following steps:

-   -   1. Preparation of the aqueous phase    -   2. Preparation of the oil phase    -   3. Preparation of the W/O emulsion (mixing the aqueous and the        oil phase)    -   4. Polymerization

After the polymer synthesis, the polymer work-up is performed by washingsteps to remove the oil phase from the polymer particles before thepolymer continues into a diminution process by mechanical means in abead mill.

Re 1. Preparations of the Aqueous Phase

The aqueous phase contains:

Compound name cas# Amount (g) H₂O 7732-18-5 56.011 DAP (1,4diacryloylpiperazine) 6342-17-2 48.535 MAA (methacrylic acid) 79-41-42.814 APS (ammonium persulfate) 7727-54-0 0.562 Gly-Phe (H-Gly-L-Phe-OH)3321-03-7 3.755

Water and DAP was mixed to a macroscopically homogenous solution bysimple stirring. Then MAA and Gly-Phe was added and dissolved at roomtemperature with stirring and ultrasound. The solution was filteredthrough a 1 μm filter. Lastly, the APS was added to the filteredsolution (99.173 g) and stirred until completely solubilized, i.e. forabout 4 minutes.

Re 2. Preparation of the Oil Phase

139.98 g of isoparaffinic hydrocarbon solvent (Isopar M®, cas#64742-47-8) and 1.068 g surfactant (Hypermer® 6212, Croda Ibérica SA)were mixed and the surfactant allowed to fully dissolve by stirring for5-10 minutes.

Ad 3. Preparation of the W/O Emulsion

60 g of the aqueous phase was added to the oil phase (and sank to thebottom) in a cylinder glass and was then subjected to high shearstirring with an ultraturrax disperser (Heidolph Diax 900 at level 5)for 3 minutes after which a stable water-in-oil (W/O) emulsion wasformed.

Ad 4. Initiation of the Polymerization

The emulsion was transferred to a two-necked reaction flask and degassedto a scheme of 3 iterations of vacuum (−0.8 bar vacuum) for 3 mins andargon flushing for 3 mins under stirring. Once degassed, 0.064 g TEMED(tetramethylethylene diamine, cas #110-18-9) was added and the emulsionheated to 70° C. in a water bath going from room temperature to 70° C.over a period of approximately 20-30 min. The resulting polymer (>90%)was in the form of 1-3 μm, regular spherical particles. They weresubsequently harvested by means of centrifugation (filtration or similarmethods are equally well suited) and washed with ethanol and water usingtangential flow filtration (TFF).

Work Up and Test of Polymer Particles from Reverse Phase EmulsionPolymerization.

The washed polymer beads were comminuted by wet bead milling using aNetzsch Labstar® equipped with a ceramic (ZrO₂) micro-chamber in twosteps: first with 0.2 mm ZrO₂ beads followed by 0.1 mm ZrO₂ beads. Theliquid phase was 50% ethanol in water adjusted to pH 10.0 with NaOH.After the final run, the average particle size was 170 nm determined bydynamic light scattering (Malvern Nanosizer®). The nanoparticles werewashed using TFF and applied to an affinity chromatography columncoupled with L-phenylalanine to test the phenylalanine bindingproperties. The nanoparticles from the reverse phase emulsionpolymerization behaved similar to nanoparticles synthesized by bulkpolymerization on the affinity chromatography.

1. A method for the preparation of molecular imprinted polymers (MIPs), which specifically bind L-phenylalanine (Phe) and L-Phe residues, said method comprising the steps of a) polymerization of a mixture comprising 2-methylprop-2-enoic acid (MAA), 1,4-bis(acryloyl)piperazine (DAP), and a template molecule consisting of L-Phe or a L-Phe derivative exposing a phenylalanine motif in the presence of a catalyst and an oxidizing agent, so as to obtain a cross-linked imprinted polymer, b) if necessary subsequently fragmenting the cross-linked imprinted polymer to obtain a first fragmented polymer, and collecting the MIPs having particle sizes smaller than 63 m, c) optionally washing and drying the polymer fraction obtained from step b), d) fragmenting the polymer fraction obtained from step b) or c) and collecting a second fragmented polymer having particle sizes in the range 150-250 nm, e) subjecting the second fragmented polymer obtained from step d to affinity chromatography where Phe constitutes the affinity tag in a chromatographic matrix, and f) recovering MIPs binding to Phe in step e).
 2. The method according to claim 1, wherein the polymerization mixture in step a contains MAA and DAP in a molar ratio of 5-30.
 3. The method according to claim 1, wherein the polymerization mixture in step a contains MAA and template molecule in a molar ratio of between 1.0-4.0, such as 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, and about 4.0.
 4. The method according to claim 1, wherein the template molecule is L-Phe derivative in the form of a peptide containing at least one L-Phe residue
 5. The method according to claim 4, wherein the templated molecule is a dipeptide or a tripeptide.
 6. The method according to claim 5, wherein the template molecule is selected from the group consisting of Gly-L-Phe, Ala-L-Phe, L-Asp-L-Phe-OMe, L-Asp-L-Phe, Gly-Gly-L-Phe, Ala-Gly-L-Phe, Gly-Ala-L-Phe, L-Phe-Gly, L-Phe-Ala, L-Phe-L-Phe, L-Phe-Ala-L-Phe, and L-Phe-Gly-L-Phe.
 7. The method according to claim 6, wherein the template molecule is Gly-L-Phe.
 8. The method according to claim 1, wherein the catalyst in step a) is selected from the group consisting of tetramethylethylenediamine, dimethylpiperazine, preferably tetramethyl ethylenediamine (TEMED).
 9. The method according to claim 1, wherein the oxidizing agent in step a) is selected from the group consisting of ammonium persulfate, potassium persulfate, and sodium thiosulfate, preferably ammonium persulfate (APS).
 10. The method according to claim 1, wherein fragmentation in step b) comprises grinding, milling, explosion, hammering, ball milling, cryo grinding, or collision homogenisation.
 11. The method according to claim 1, wherein the MIPs collected in step b) have sizes in the 25-63 μm range.
 12. The method according to claim 1, wherein step b) entails collection of MIPs able to pass through a sieve with a 63 μm cut-off.
 13. The method according claim 12, wherein step b) further entails collection of MIPs that are retained on a sieve with a 25 μm cut-off.
 14. The method according to claim 1, wherein washing is performed at alternating pH with an organic solvent.
 15. The method according to claim 1, wherein the MIPs are packed in an HPLC column in step c) during washing under elevated pressure.
 16. The method according to claim 1, wherein fragmentation in step d) is carried out in a ball mill or bead mill.
 17. The method according to claim 1, wherein collection of MIPs in step d) is carried out by suspension of MIPs in an aqueous solvent such as water, incubation in an ultrasound bath, centrifugation, and isolation of the supernatant, which contains the 150-250 nm MIPs.
 18. The method according to claim 1, wherein step d) entails washing to separate the MIPs from residual template molecule, such as a dialytic washing step or a washing step in an ultracentrifuge.
 19. The method according to claim 1 wherein the affinity chromatography in step e) is carried out on a packed bed chromatographic column using a stationary chromatographic matrix, and where the MIPs are suspended in a buffered aqueous solvent.
 20. The method according to claim 1, wherein recovery in step f) comprises elution of the MIPs from the chromatographic matrix.
 21. A molecular imprinted polymer (MIP), which specifically binds L-phenylalanine (Phe), wherein said MIP is comprised of polymerized methacrylic acid (MAA) cross-linked with 1,4-diacryloylpiperazine (DAP).
 22. The MIP according to claim 21, wherein the molar ratio between MAA residues and DAP residues is between 5 and
 30. 23. The MIP according to claim 21, which is obtainable or obtained by the method according claim
 1. 24. The molecular imprinted polymer according to claim 21, which has a K_(D) for binding to L-Phe of less than 10⁻⁷, such as less than 10⁻⁸, less than 9×10⁻⁹, less than 8×10⁻⁹, less than 7×10⁻⁹, less than 6×10⁻⁹, less than 5×10⁻⁹, less than 4×10⁻⁹, less than 3×10⁻⁹, and less than 2×10⁻⁹.
 25. The molecular imprinted polymer according to claim 24, where the K_(D) is about 10⁻⁹.
 26. A composition comprising molecular imprinted polymers according to claim 21, said composition comprising a pharmaceutically acceptable carrier and/or diluent and/or excipient, wherein said composition is adapted for oral administration.
 27. A method for the preparation of MIPs, which specifically bind L-Tyrosine (Tyr), said method comprising recovering MIPs that bind L-Tyrosine from an initial composition of MIPs that have been prepared using, as the template molecule during polymerization and cross-linking, a molecule comprising at least one phenylalanine motif and comprising no Tyr residues.
 28. The method according to claim 27, wherein the template molecule is as defined in claim
 1. 29. The method according to claim 27, which comprises that the initial composition of MIPs is separated in at least 2 fractions, and recovering Tyr binding MIPs from a fraction, which is essentially free from MIPs that bind the Phe-containing capture probe.
 30. The method according to claim 27, which comprises the steps of a) polymerization of a mixture comprising 2-methylprop-2-enoic acid (MAA), 1,4-bis(acryloyl)piperazine (DAP), and a template molecule defined in claim 1, in the presence of a catalyst and an oxidizing agent, so as to obtain a cross-linked imprinted polymer, b) if necessary subsequently fragmenting the cross-linked imprinted polymer to obtain a first fragmented polymer, and collecting the fraction thereof having particle sizes smaller than 63 μm, c) optionally washing and drying the polymer fraction obtained from step b), d) fragmenting the polymer fraction obtained from step c) and collecting a second fragmented polymer having particle sizes in the range 150-250 nm, and e) subjecting the second fragmented polymer obtained from step d) to affinity chromatography where e1) Tyr constitutes the affinity tag in a chromatographic matrix, or e2) Phe constitutes the affinity tag in a chromatographic matrix, recovering MIPs not binding to Phe, and subjecting the MIPs not binding to Phe to further affinity chromatography where Tyr constitutes the affinity tag in a chromatographic matrix, and f) recovering MIPs binding to Tyr in step e).
 31. A molecular imprinted polymer (MIP), which specifically binds L-tyrosine (Tyr), wherein said MIP is comprised of polymerized methacrylic acid (MAA) cross-linked with 1,4-diacryloulpiperazine (DAP).
 32. The MIP according to claim 31, wherein the molar ratio between MAA residues and DAP residues is between 5 and
 30. 33. The MIP according to claim 31, which is obtainable or obtained by the method according to claim
 27. 34. The molecular imprinted polymer according to claim 31, which has a K_(D) for binding to Tyr of less than 10⁻⁷, such as less than 10⁻⁸, less than 9×10⁻⁹, less than 8×10⁻⁹, less than 7×10⁻⁹, less than 6×10⁻⁹, less than 5×10⁻⁹, less than 4×10⁻⁹, less than 3×10⁻⁹, or less than 2×10⁻⁹.
 35. The molecular imprinted polymer according to claim 34, where the K_(D) is about 10⁻⁹.
 36. A composition comprising molecular imprinted polymers according to claim 31, said composition comprising a pharmaceutically acceptable carrier and/or diluent and/or excipient, wherein said composition is adapted for oral administration.
 37. A method of treatment of phenylketonuria in a person in need thereof, said method comprising administering to a person in need thereof the MIPs according to claim 21 or the composition according to claim 26 so as to deliver a daily effective dose of MIPs according to claim
 21. 38. A method for treatment of tyrosineamia and/or alkaptonuria, the method comprising administering to a person in need thereof 1) MIPs that bind phenylalanine, preferably the MIPs according to claim 21 or the composition according to claim 26 so as to deliver a daily effective dose of MIPs according to claim 21; and/or 2) MIPs that bind tyrosine according to claim 31 or the composition according to claim 36, so as to deliver a daily effective dose of MIPs according to claim
 31. 39. The method according to claim 38, wherein the treatment is carried out as an adjuvant therapy to nitisinone treatment of alkaptonuria or tyrosinemia-I.
 40. The method according to claim 37, wherein the daily effective dose is 1-35 g per 70 kg body weight.
 41. The method according to claim 40, wherein the effective dose per meal is at most or about 34 g/70 kg, such as at most or about 33, at most or about 32, at most or about 31, at most or about 30, at most or about 29, at most or about 28, at most or about 27, at most or about 26, at most or about 25, at most or about 24 at most or about 23, at most or about 22, at most or about 21, at most or about 20, at most or about 19, at most or about 18, at most or about 17, at most or about 16, at most or about 15, at most or about 14, at most or about 13, at most or about 12, at most or about 11, at most or about 10, at most or about 9, at most or about 8, at most or about 7, at most or about 6, at most or about 5, at most or about 4, at most or about 3, at most or about 3, and at most or about 2 g/70 kg.
 42. The method according to claim 40, wherein daily effective dose is 20-45 g/70 kg, such as 25-40 g/70 kg, and 30-35 g/70 kg.
 43. The method according to claim 40, wherein daily effective dose is about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, or about 105 g per 70 kg body weight. 